Differences in the anatomy and physiology of the human and rat respiratory tracts and impact on toxicological assessments.

Inhalation is a critical route through which substances can exert adverse effects in humans; therefore, it is important to characterize the potential effects that inhaled substances may have on the human respiratory tract by using fit for purpose, reliable, and human relevant testing tools. In regulatory toxicology testing, rats have primarily been used to assess the effects of inhaled substances as they-being mammals-share similarities in structure and function of the respiratory tract with humans. However, questions about inter-species differences impacting the predictability of human effects have surfaced. Disparities in macroscopic anatomy, microscopic anatomy, or physiology, such as breathing mode (e.g., nose-only versus oronasal breathing), airway structure (e.g., complexity of the nasal turbinates), cell types and location within the respiratory tract, and local metabolism may impact inhalation toxicity testing results. This review shows that these key differences describe uncertainty in the use of rat data to predict human effects and supports an opportunity to harness modern toxicology tools and a detailed understanding of the human respiratory tract to develop testing approaches grounded in human biology. Ultimately, as the regulatory purpose is protecting human health, there is a need for testing approaches based on human biology and mechanisms of toxicity.

Defined approaches to classify agrochemical formulations into EPA hazard categories developed using EpiOcularTM reconstructed human corneal epithelium and bovine corneal opacity and permeability assays.

Many sectors have seen complete replacement of the in vivo rabbit eye test with reproducible and relevant in vitro and ex vivo methods to assess the eye corrosion/irritation potential of chemicals. However, the in vivo rabbit eye test remains the standard test used for agrochemical formulations in some countries. Therefore, two defined approaches (DAs) for assessing conventional agrochemical formulations were developed, using the EpiOcularTM Eye Irritation Test (EIT) [Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 492] and the Bovine Corneal Opacity and Permeability (OECD TG 437; BCOP) test with histopathology. Presented here are the results from testing 29 agrochemical formulations, which were evaluated against the United States Environmental Protection Agency's (EPA) pesticide classification system, and assessed using orthogonal validation, rather than direct concordance analysis with the historical in vivo rabbit eye data. Scientific confidence was established by evaluating the methods and testing results using an established framework that considers fitness for purpose, human biological relevance, technical characterisation, data integrity and transparency, and independent review. The in vitro and ex vivo methods used in the DAs were demonstrated to be as or more fit for purpose, reliable and relevant than the in vivo rabbit eye test. Overall, there is high scientific confidence in the use of these DAs for assessing the eye corrosion/irritation potential of agrochemical formulations.

Human cell-based in vitro systems to assess respiratory toxicity: a case study using silanes.

Inhalation is a major route by which human exposure to substances can occur. Resources have therefore been dedicated to optimize human-relevant in vitro approaches that can accurately and efficiently predict the toxicity of inhaled chemicals for robust risk assessment and management. In this study-the IN vitro Systems to PredIct REspiratory toxicity Initiative-2 cell-based systems were used to predict the ability of chemicals to cause portal-of-entry effects on the human respiratory tract. A human bronchial epithelial cell line (BEAS-2B) and a reconstructed human tissue model (MucilAir, Epithelix) were exposed to triethoxysilane (TES) and trimethoxysilane (TMS) as vapor (mixed with N2 gas) at the air-liquid interface. Cell viability, cytotoxicity, and secretion of inflammatory markers were assessed in both cell systems and, for MucilAir tissues, morphology, barrier integrity, cilia beating frequency, and recovery after 7 days were also examined. The results show that both cell systems provide valuable information; the BEAS-2B cells were more sensitive in terms of cell viability and inflammatory markers, whereas MucilAir tissues allowed for the assessment of additional cellular effects and time points. As a proof of concept, the data were also used to calculate human equivalent concentrations. As expected, based on chemical properties and existing data, the silanes demonstrated toxicity in both systems with TMS being generally more toxic than TES. Overall, the results demonstrate that these in vitro test systems can provide valuable information relevant to predicting the likelihood of toxicity following inhalation exposure to chemicals in humans.

Cryopreserved human precision-cut lung slices provide an immune competent pulmonary test system for "on-demand" use and long-term cultures.

Human precision-cut lung slices (hPCLS), considered a highly relevant ex vivo model of the lung, offer native architecture and cells of the lung tissue including respiratory parenchyma, small airways, and immune competent cells. However, the irregular availability of donor lungs has limited the accessibility of this system. As described here, thousands of hPCLS can be created from 1 lung, cryopreserved, and used "on demand" by applying slicing and cryopreservation methodology improvements. Fresh and cryopreserved (∼7 and ∼34 weeks; F&C) hPCLS from 1 donor lung were cultured for up to 29 days and evaluated for biomass, viability, tissue integrity, and inflammatory markers in response to lipopolysaccharide (LPS; 5 µg/ml) and Triton X-100 (TX100; 0.1%) challenge (24 h) at days 1, 8, 15, 22, and 29 following culture initiation. The F&C hPCLS retained biomass, viability, and tissue integrity throughout the 29 days and demonstrated immune responsiveness with up to ∼30-fold LPS-induced cytokine increases. Histologically, more than 70% of normal cytomorphological features were preserved in all groups through day 29. Similar retention of tissue viability and immune responsiveness post cryopreservation (4-6 weeks) and culture (up to 14 days) was observed in hPCLS from additional 3 donor lungs. Banking cryopreserved hPCLS from various donors (and disease states) provides a critical element in researching human-derived pulmonary tissue. The retention of viability and functional responsiveness (≥4 weeks) allows evaluation of long-term, complex endpoints reflecting key events in Adverse Outcome Pathways and positions hPCLS as a valuable human-relevant model for use in regulatory applications.

Use of new approach methodologies (NAMs) to meet regulatory requirements for the assessment of tobacco and other nicotine-containing products.

Regulatory frameworks on tobacco and other nicotine-containing products (TNCP) continue to evolve as novel products emerge, including electronic nicotine delivery systems (e.g., electronic cigarettes or vaping products), heated tobacco products, or certain smokeless products (e.g., nicotine pouches). This article focuses on selected regulations for TNCPs that do not make health claims, and on the opportunities to use new approach methodologies (NAMs) to meet regulatory requirements for toxicological information. The manuscript presents a brief overview of regulations and examples of feedback from regulatory agencies whilst highlighting NAMs that have been successfully applied, or could be used, in a regulatory setting, either as stand-alone methods or as part of a weight-of-evidence approach to address selected endpoints. The authors highlight the need for agencies and stakeholders to collaborate and communicate on the development and application of NAMs to address specific regulatory toxicological endpoints. Collaboration across sectors and geographies will facilitate harmonized use of robust testing approaches to evaluate TNCPs without animal testing.

Use of new approach methodologies (NAMs) to meet regulatory requirements for the assessment of industrial chemicals and pesticides for effects on human health.

New approach methodologies (NAMs) are increasingly being used for regulatory decision making by agencies worldwide because of their potential to reliably and efficiently produce information that is fit for purpose while reducing animal use. This article summarizes the ability to use NAMs for the assessment of human health effects of industrial chemicals and pesticides within the United States, Canada, and European Union regulatory frameworks. While all regulations include some flexibility to allow for the use of NAMs, the implementation of this flexibility varies across product type and regulatory scheme. This article provides an overview of various agencies' guidelines and strategic plans on the use of NAMs, and specific examples of the successful application of NAMs to meet regulatory requirements. It also summarizes intra- and inter-agency collaborations that strengthen scientific, regulatory, and public confidence in NAMs, thereby fostering their global use as reliable and relevant tools for toxicological evaluations. Ultimately, understanding the current regulatory landscape helps inform the scientific community on the steps needed to further advance timely uptake of approaches that best protect human health and the environment.

A framework for establishing scientific confidence in new approach methodologies.

Robust and efficient processes are needed to establish scientific confidence in new approach methodologies (NAMs) if they are to be considered for regulatory applications. NAMs need to be fit for purpose, reliable and, for the assessment of human health effects, provide information relevant to human biology. They must also be independently reviewed and transparently communicated. Ideally, NAM developers should communicate with stakeholders such as regulators and industry to identify the question(s), and specified purpose that the NAM is intended to address, and the context in which it will be used. Assessment of the biological relevance of the NAM should focus on its alignment with human biology, mechanistic understanding, and ability to provide information that leads to health protective decisions, rather than solely comparing NAM-based chemical testing results with those from traditional animal test methods. However, when NAM results are compared to historical animal test results, the variability observed within animal test method results should be used to inform performance benchmarks. Building on previous efforts, this paper proposes a framework comprising five essential elements to establish scientific confidence in NAMs for regulatory use: fitness for purpose, human biological relevance, technical characterization, data integrity and transparency, and independent review. Universal uptake of this framework would facilitate the timely development and use of NAMs by the international community. While this paper focuses on NAMs for assessing human health effects of pesticides and industrial chemicals, many of the suggested elements are expected to apply to other types of chemicals and to ecotoxicological effect assessments.

Maximizing the relevance and reproducibility of A549 cell culture using FBS-free media.

Scientists are using in vitro methods to answer important research questions and implementing strategies to maximize the reliability and human relevance of these methods. One strategy is to replace the use of fetal bovine serum (FBS)-an undefined and variable mixture of biomolecules-in cell culture media with chemically defined or xeno-free medium. In this study, A549 cells, a human lung alveolar-like cell line commonly used in respiratory research, were transitioned from a culture medium containing FBS to media without FBS. A successful transition was determined based on analysis of cell morphology and functionality. Following transition to commercially available CnT-Prime Airway (CELLnTEC) or X-VIVO™ 10 (Lonza) medium, the cells were characterized by microscopic evaluation and calculation of doubling time. Their genotype, morphology, and functionality were assessed by monitoring the expression of gene markers for lung cell types, surfactant production, cytokine release, the presence of multilamellar bodies, and cell viability following sodium dodecyl sulphate exposure. Our results showed that A549 cells successfully transitioned to FBS-free media under submerged and air-liquid-interface conditions. Cells grown in X-VIVO™ 10 medium mimicked cellular characteristics of FBS-supplemented media while those grown in CnT-Prime Airway medium demonstrated characteristics possibly more reflective of normal human alveolar epithelial cells.

Replacement of in vivo leptospirosis vaccine potency testing in the United States.

The U.S. Department of Agriculture (USDA) regulates the potency testing of leptospirosis vaccines, which are administered to animals to protect against infection by Leptospira bacteria. Despite the long-term availability of in vitro test methods for assessing batch potency, the use of hamsters in lethal in vivo batch potency testing persists to varying degrees across leptospirosis vaccine manufacturers. For all manufacturers of these products, data collected from public USDA records show an estimated 40% decline in the annual use of hamsters from 2014 to 2020, with an estimated 55% decrease in the number of hamsters expected to have been used in leptospirosis vaccine potency tests (i.e., those in USDA Category E). An estimated 49,000 hamsters were used in 2020, with about 15,000 hamsters in Category E specifically. Based on this assessment, additional efforts are needed to fully implement in vitro batch potency testing as a replacement for the in vivo batch potency test. We propose steps that can be taken collaboratively by the USDA Center for Veterinary Biologics (CVB), manufacturers of leptospirosis vaccines, government agencies, and non-governmental organizations to accelerate broader use of the in vitro approach.

Rethinking chronic toxicity and carcinogenicity assessment for agrochemicals project (ReCAAP): A reporting framework to support a weight of evidence safety assessment without long-term rodent bioassays.

Rodent cancer bioassays have been long-required studies for regulatory assessment of human cancer hazard and risk. These studies use hundreds of animals, are resource intensive, and certain aspects of these studies have limited human relevance. The past 10 years have seen an exponential growth of new technologies with the potential to effectively evaluate human cancer hazard and risk while reducing, refining, or replacing animal use. To streamline and facilitate uptake of new technologies, a workgroup comprised of scientists from government, academia, non-governmental organizations, and industry stakeholders developed a framework for waiver rationales of rodent cancer bioassays for consideration in agrochemical safety assessment. The workgroup used an iterative approach, incorporating regulatory agency feedback, and identifying critical information to be considered in a risk assessment-based weight of evidence determination of the need for rodent cancer bioassays. The reporting framework described herein was developed to support a chronic toxicity and carcinogenicity study waiver rationale, which includes information on use pattern(s), exposure scenario(s), pesticidal mode-of-action, physicochemical properties, metabolism, toxicokinetics, toxicological data including mechanistic data, and chemical read-across from similar registered pesticides. The framework could also be applied to endpoints other than chronic toxicity and carcinogenicity, and for chemicals other than agrochemicals.

Use of Cause-and-Effect Analysis to Optimize the Reliability of In Vitro Inhalation Toxicity Measurements Using an Air-Liquid Interface.

In vitro inhalation toxicology methods are increasingly being used for research and regulatory purposes. Although the opportunity for increased human relevance of in vitro inhalation methods compared to in vivo tests has been established and discussed, how to systematically account for variability and maximize the reliability of these in vitro methods, especially for assays that use cells cultured at an air-liquid interface (ALI), has received less attention. One tool that has been used to evaluate the robustness of in vitro test methods is cause-and-effect (C&E) analysis, a conceptual approach to analyze key sources of potential variability in a test method. These sources of variability can then be evaluated using robustness testing and potentially incorporated into in-process control measurements in the assay protocol. There are many differences among in vitro inhalation test methods including the use of different types of biological test systems, exposure platforms/conditions, substances tested, and end points, which represent a major challenge for use in regulatory testing. In this manuscript, we describe how C&E analysis can be applied using a modular approach based on the idea that shared components of different test methods (e.g., the same exposure system is used) have similar sources of variability even though other components may differ. C&E analyses of different in vitro inhalation methods revealed a common set of recommended exposure systems and biological in-process control measurements. The approach described here, when applied in conjunction with Good Laboratory Practices (GLP) criteria, should help improve the inter- and intralaboratory agreement of in vitro inhalation test results, leading to increased confidence in these methods for regulatory and research purposes.

Human-relevant approaches to assess eye corrosion/irritation potential of agrochemical formulations.

There are multiple in vitro and ex vivo eye irritation and corrosion test methods that are available as internationally harmonized test guidelines for regulatory use. Despite their demonstrated usefulness to a broad range of substances through inter-laboratory validation studies, they have not been widely adopted for testing agrochemical formulations due to a lack of concordance with parallel results from the traditional regulatory test method for this endpoint, the rabbit eye test. The inherent variability of the rabbit test, differences in the anatomy of the rabbit and human eyes, and differences in modelling exposures in rabbit eyes relative to human eyes contribute to this lack of concordance. Ultimately, the regulatory purpose for these tests is protection of human health, and, thus, there is a need for a testing approach based on human biology. This paper reviews the available in vivo, in vitro and ex vivo test methods with respect to their relevance to human ocular anatomy, anticipated exposure scenarios, and the mechanisms of eye irritation/corrosion in humans. Each of the in vitro and ex vivo methods described is generally appropriate for identifying non-irritants. To discriminate among eye irritants, the human three-dimensional epithelial and full thickness corneal models provide the most detailed information about the severity of irritation. Consideration of the mechanisms of eye irritation, and the strengths and limitations of the in vivo, in vitro and ex vivo test methods, show that the in vitro/ex vivo methods are as or more reflective of human biology and less variable than the currently used rabbit approach. Suggestions are made for further optimizing the most promising methods to distinguish between severe (corrosive), moderate, mild and non-irritants and provide information about the reversibility of effects. Also considered is the utility of including additional information (e.g. physical chemical properties), consistent with the Organization for Economic Cooperation and Development's guidance document on an integrated approach to testing and assessment of potential eye irritation. Combining structural and functional information about a test substance with test results from human-relevant methods will ensure the best protection of humans following accidental eye exposure to agrochemicals.

Using the monocyte activation test as a stand-alone release test for medical devices.

Monocyte activation tests (MAT) are widely available but rarely used in place of animal-based pyrogen tests for safety assessment of medical devices. To address this issue, the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods and the PETA International Science Consortium Ltd. convened a workshop at the National Institutes of Health on September 18-19, 2018. Participants included representatives from MAT testing laboratories, medical device manufacturers, the U.S. Food and Drug Administration's Center for Devices and Radiologic Health (CDRH), the U.S. Pharmacopeia, the International Organization for Standardization, and experts in the development of MAT protocols. Discussions covered industry experiences with the MAT, remaining challenges, and how CDRH's Medical Device Development Tools (MDDT) Program, which qualifies tools for use in evaluating medical devices to streamline device development and regulatory evaluation, could be a pathway to qualify the use of MAT in place of the rabbit pyrogen test and the limulus amebocyte lysate test for medical device testing. Workshop outcomes and follow-up activities are discussed.

Mind the gaps: Prioritizing activities to meet regulatory needs for acute systemic lethality

Efforts are underway to develop and implement nonanimal approaches which can characterize acute systemic lethality. A workshop was held in October 2019 to discuss developments in the prediction of acute oral lethality for chemicals and mixtures, as well as progress and needs in the understanding and modeling of mechanisms of acute lethality. During the workshop, each speaker led the group through a series of charge questions to determine clear next steps to progress the aims of the workshop. Participants concluded that a variety of approaches will be needed and should be applied in a tiered fashion. Non-testing approaches, including waiving tests, computational models for single chemicals, and calculating the acute lethality of mixtures based on the LD50 values of mixture components, could be used for some assessments now, especially in the very toxic or non-toxic classification ranges. Agencies can develop policies indicating contexts under which mathematical approaches for mixtures assessment are acceptable; to expand applicability, poorly predicted mixtures should be examined to understand discrepancies and adapt the approach. Transparency and an understanding of the variability of in vivo approaches are crucial to facilitate regulatory application of new approaches. In a replacement strategy, mechanistically based in vitro or in silico models will be needed to support non-testing approaches especially for highly acutely toxic chemicals. The workshop discussed approaches that can be used in the immediate or near term for some applications and identified remaining actions needed to implement approaches to fully replace the use of animals for acute systemic toxicity testing.

Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes.

Expansion in production and commercial use of nanomaterials increases the potential human exposure during the lifecycle of these materials (production, use, and disposal). Inhalation is a primary route of exposure to nanomaterials; therefore it is critical to assess their potential respiratory hazard. Herein, we developed a three-dimensional alveolar model (EpiAlveolar) consisting of human primary alveolar epithelial cells, fibroblasts, and endothelial cells, with or without macrophages for predicting long-term responses to aerosols. Following thorough characterization of the model, proinflammatory and profibrotic responses based on the adverse outcome pathway concept for lung fibrosis were assessed upon repeated subchronic exposures (up to 21 days) to two types of multiwalled carbon nanotubes (MWCNTs) and silica quartz particles. We simulate occupational exposure doses for the MWCNTs (1-30 µg/cm2) using an air-liquid interface exposure device (VITROCELL Cloud) with repeated exposures over 3 weeks. Specific key events leading to lung fibrosis, such as barrier integrity and release of proinflammatory and profibrotic markers, show the responsiveness of the model. Nanocyl induced, in general, a less pronounced reaction than Mitsui-7, and the cultures with human monocyte-derived macrophages (MDMs) showed the proinflammatory response at later time points than those without MDMs. In conclusion, we present a robust alveolar model to predict inflammatory and fibrotic responses upon exposure to MWCNTs.

Evaluation of the avian acute oral and sub-acute dietary toxicity test for pesticide registration.

The United States Environmental Protection Agency (USEPA), as well as other international regulatory agencies, require pesticide registrants to submit toxicity data that are used to conduct ecological risk assessments. While the USEPA has required both an acute oral and sub-acute dietary test in birds, trends in the use of data from these tests over the past 20 years have suggested that the avian sub-acute dietary test generally does not contribute to risk assessment conclusions. To address this question, a retrospective analysis was conducted to evaluate 119 pesticides with publicly available ecological risk assessments that were registered into commerce between 1998 and 2017. New pesticides (i.e., registered in the United States within the past 20 years) were chosen for the retrospective analysis to show utility of these tests for modern pesticide chemistries. Risk quotient (RQ) values (a point estimate of exposure divided by a deterministic toxicity endpoint) from the avian acute oral and dietary tests, as well as risk assessment conclusions, were compared to determine which test(s) drove the risk assessment findings. The RQ values were chosen as the data point for comparison in order to assess total risk (i.e., exposure and toxicity). After comparing RQ values from avian acute oral versus sub-acute dietary tests, there was only one case in which an avian sub-acute dietary RQ was greater than the acute oral RQ. Thus, the sub-acute dietary test did not identify risk in greater than 99% (118 out of 119) of chemicals based on results that either the acute oral RQ was higher than the sub-acute dietary RQ, or both the acute oral and the subacute dietary tests did not generate an RQ value of concern. For the one exception, both the oral and sub-acute RQ values were greater than the USEPA's level of concern for endangered species. Based on the results of the retrospective analysis, it is concluded that in most cases avian risk can confidently be assessed without conducting the sub-acute dietary test.

Pathway-based predictive approaches for non-animal assessment of acute inhalation toxicity.

New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity. This paper describes the AOPs and the toolbox of in vitro and in silico models that can be used to assess the key events leading to toxicity following inhalation exposure. Because the optimal testing strategy will vary depending on the substance of interest, here we present a decision tree approach to identify an appropriate non-animal integrated testing strategy that incorporates consideration of a substance's physicochemical properties, relevant mechanisms of toxicity, and available in silico models and in vitro test methods. This decision tree can facilitate standardization of the testing approaches. Case study examples are presented to provide a basis for proof-of-concept testing to illustrate the utility of non-animal approaches to inform hazard identification and risk assessment of humans exposed to inhaled substances.